DNA条形码在鲱形目鱼类物种鉴定和系统进化分析中的应用

Species identification and phylogenetic relationships in order Clupeiformes based on DNA barcoding

采用PCR特异性扩增获得中国近海鲱形目(Clupeiformes)2科6属7种的48条线粒体COI基因序列,结合从Gen Bank筛选出的4科40属83种的COI基因序列225条,对鲱形目鱼类的COI条形码基因特征、种内与种间遗传距离及其分子系统进化关系进行了分析,探索了DNA条形码技术在辅助鱼类物种鉴定和分类中的适应性。结果表明,4科41属90种273条COI基因序列的平均碱基组成为T:28.3%、C:28.3%、A:24.2%、G:19.2%,碱基组成表现出明显偏倚性。鲱形目鱼类种间的平均遗传距离为0.131,种内平均遗传距离为0.003,种间距离为种内距离的41倍;系统学分析结果显示,97.8%的鱼类在系统进化树上均为单系。可见,鲱形目鱼类DNA条形码符合物种鉴定的要求,且基于COI基因所建的NJ树对物种分类具有较为准确的辨识力。系统进化分析结果表明,COI基因不仅能够解决低阶分类单元的系统进化关系,对于高阶分类单元的系统分析研究结果也有一定参考价值。

The order Clupeiformesis in the Class Osteichthyes, and many of these species are of significant economic value. The mitochondrial cytochrome c oxidase subunit 1（COI） gene sequences of seven species pertaining to six genera in Clupeiformes collected from Chinese coastal waters were amplified using a polymerase chain reaction technique. The homologous sequences of other species of Clupeiformes from Gen Bank were also included in this study. A total of 273 single sequences from 90 species pertaining to 41 genera in Clupeiformes were barcoded using the COI gene and then sequenced and compared to verify the feasibility of using DNA barcoding to identify species. Clustal X and MEGA6.0 were used to calculate base composition, sequence substitutions, and genetic distance. The results indicated that mean T, C, A, and G contents were 28.3%, 28.3%, 24.2%, and 19.2%, respectively, indicating that base composition was biased.The nucleotide sequences revealed base substitutions, including 71 conversion sites and 46 transversion sites. Most appeared on the third codon, including 62 conversion sites and 45 transversion sites. The second codon did not have conversions or transversions. The conversion and transversion ratio reflects evolutionary rate; if the probability of transversion is greater, the conversion and transversion ratio is smaller, and the evolutionary rate is faster. The evolutionary rate of the third codon was the fastest, followed by the first codon, and the second codon was the most stable. DNA barcoding can be used to identify species if the distance between congeners is much larger than that between conspecifics and different individuals of the same species are clustered in the same phylogenetic tree branch. The congeneric and conspecificgenetic distances were 0.131 and 0.003, respectively, and the congeneric distance was 41 times that of the conspecific distance. However, some congeneric distances were too small or the conspecific distances were too large, leading to different names for the same species and intraspecific differentiation. All conspecifics clustered together in the phylogenetic tree, except Dorosoma cepedianum and Dorosoma anale, suggesting that these may be the same species. The phylogenetic analysis showed that 97.8% of the fish were monophyletic. In summary, the DNA barcodes of clupeiform fish complied with the requirements for species identification, and the neighbor-joining tree based on the COI gene identified species more accurately. The phylogenetic results show that the COI gene was suitable for clarifying primary taxa and provided some reference value for the phylogenetic relationships of senior taxa.